A common precursor for the hematopoietic and endothelial cell lineages, termed the hemangioblast, was first proposed many years ago. Recent studies have demonstrated that embryoid bodies (EBs) generated from embryonic stem (ES) cells differentiated in culture contain a novel VEGF-responsive precursor with the properties of the hemangioblast. These precursors, in response to VEGF, generate blast cell colonies in methylcellulose cultures that contain both hematopoietic and endothelial precursors. We have recently identified an earlier precursor population from early EBs. These cells generate morphologically distinct colonies (transitional colonies) that based upon gene expression analyses and developmental potential in culture are not as developmentally advanced as the blast colony. Blast colonies are postmesodermal and express markers for both the hematopoietic and endothelial lineages, whereas the transitional colonies express mesodermal markers in addition to early hematopoietic and endothelial markers. An individual transitional colony then contains populations of cells undergoing mesodermal commitment, presumably through the bi-potential hemangioblast, to the hematopoietic and endothelial lineages. Subtractive hybridization has been carried out between a single EB which has undergone mesoderm induction and a single transitional colony which has proceeded to early hematopoietic and endothelial lineage differentiation. The subtraction has enriched for transcripts expressed specifically, or at higher levels, in the transitional colony which should include genes involved in hemangioblast commitment and early hematopoietic and endothelial differentiation. The first aim of this proposal is to identify,transcripts within this subtraction library that play a potential role in this developmental program. The second aim involves looking at selected candidates via functional assays using the ES/EB differentiation system which will allow a quantitative measure of the hemangioblast and hematopoietic precursors. The information obtained from these experiments will provide the first molecular characterization of the ES cell-derived hemangioblast and will help to define the events involved in its development, growth and differentiation. It is hoped that these results will also lead to a better understanding of the relationship of the hemangioblast to other early embryonic hematopoietic precursors, including the long-term repopulating stem cell. Finally, defining the molecular events that control the growth and differentiation of this novel embryonic precursor could provide new insights into the underlying bases of abnormal hematopoietic development and blood cell diseases in man.